In performing analytical reactions, e.g., analyzing chemical, biochemical, biological or other reactions, a common difficulty is maximizing the signal to noise ratio, or the ratio of relevant detected events from irrelevant or less relevant detected events that are indistinguishable from or otherwise interfere with the relevant detected events. The less relevant detected events may derive from a variety of sources, including, e.g., ambient concentrations of reactants, built up detectable products, interfering signals from other unrelated components of the analysis, e.g., solution contents, reaction vessel interference, and even instrument originating background signal levels. One major source of noise in many analytical reactions comes from the non-specific association of signal producing reaction components with components of the reaction of interest. This includes, for example, association of labeled reaction components, products etc., with solid phase components of an analytical reaction, e.g., beads, surfaces, or the like, as well as association with other reaction components, e.g., enzymes or other proteins, nucleic acids, cells, or the like.
In addition to reduction of background noise, enzymatic reactions suffer from aberrant enzyme behavior, e.g., changes in catalytic rate, pausing, dissociating, premature termination, changes in error profile, reduced rates of substrate binding and/or translocation, and the like. For example, in polymerase-mediated sequencing-by-synthesis reactions, polymerases have been observed to pause for extended periods of time before resuming normal nucleotide incorporation. In some instances, polymerase kinetics change depending on the type of nucleotide analog being incorporated. For example, some nucleotide analogs are incorporated at a slower rate than other nucleotide analogs, or the incorporation rate is highly variable during the course of a reaction. These behaviors have also been associated with shortened readlength and a worsening of other measures of sequencing performance, e.g., error metrics and overall accuracy. As such, preventing or reducing aberrant enzyme behavior in enzyme-catalyzed analytical reactions would be beneficial, e.g., to enhance enzyme performance and improve data generation.
Accordingly, it is desirable to reduce the overall level of background noise in analytical reactions, and particularly to reduce such background that derives from non-specific association of detectable reaction components with other components of the analysis. It is also desirable to increase or enhance the fluorescence intensity and/or improve fluorophore photostability. Yet further, it is desirable to prevent or reduce aberrant enzyme behavior, e.g., in order to increase readlength and accuracy of analytical reactions such as polymerase-mediated single molecule sequencing. The present invention provides solutions to these and other problems.